This invention relates to nucleotide sequences encoding fructan synthesizing enzymes, a recombinant DNA sequence comprising one or more of said nucleotide sequences, a method for producing a genetically transformed host organism showing a modified fructan profile, and transformed plants or plant parts showing said modified fructan profile.
Fructans refer to a group of carbohydrate compounds in which one or more fructosyl-fructose linkages constitute a majority of the linkages. Fructans are fructosepolymers with usually, but not necessarily, one terminal glucosylunit [G-(F).sub.n, G=optional, n.gtoreq.2]. The fructosyl-fructose linkages in fructans are of the .beta.-2,6 or .beta.-2,1 linkage type. Fructans with predominantly .beta.-2,6 fructosyl-fructose linkages are usually called levan(s). Fructans with predominantly .beta.-2,1 fructosyl-fructose linkages are usually called inulin(s).
Fructan biosynthesis is common in several bacterial, fungal and algal families and also in specific plant families, such as the Liliaceae (e.g. Allium cepa), Poaceae (e.g. Lolium perenne) and Asteraceae (e.g. Helianthus tuberosus). The function of fructans in bacteria and fungi is poorly understood. It has been suggested that fructans act as extracellular stores of carbohydrate that can be mobilized during periods of carbohydrate stress (Jacques, 1993). In plants, fructans can function as reserve carbohydrates which serve as a source of carbon for (re)growth (Meier and Reid, 1982). In fructan-storing crops, tructan synthesis is restricted not only to specific organs (e.g. the stems or tubers of H. tuberosus, the bulbs of Allium spp, the leaf bases and stems of grasses) but also specific cell types within these organs (usually the parenchyma cells). In these specific cell types, the vacuole is probably the location of both fructan biosynthesis and storage (Darwen and John, 1989; Wagner et al., 1983).
In bacteria, examples of fructan synthesizing bacteria are Streptococcus mutans and Bacillus subtilus, the biosynthesis of fructans from sucrose is catalysed by only one enzyme: levansucrase (EC 2.4.1.10) in B. subtilus (Dedonder 1966) and levansucrase, but also called fructosyltransferase, (FTF, EC 2.4.1.10) in S. mutans (Carlsson, 1970). Bacterial fructan synthesis proceeds via the direct transfer of fructose from a donor-sucrose (G-F) to sucrose or other acceptor molecules according to the following reversible reaction: EQU n(G-F)+acceptor.fwdarw.n(G)+acceptor-(F).sub.u, (1)
where n may be larger than 10.000
Water, hexoses, sucrose, oligosaccharides and levan may act as acceptor molecules for fructosyl units from sucrose (fructosyl donor).
Bacterial DNA sequences encoding FTF in S. mutans and levansucrase in B. subtilus are already described in the literature (Sato and Kuramitsu, 1986; Steinmetz et al. 1985). Bacterial genes from several sources were used to transform specific host plants which normally cannot synthesize fructans, thereby inducing fructan synthesis (see for example: Van der Meer et al., 1994; Ebskamp et al., 1994). A method to enhance the solid content of tomato fruits, using the levansucrase gene from B. subtilus and the dextransucrase gene from Leuconostoc mesenretoides is described in application WO 89/12386. A method to modify the fructan pattern in plants which normally cannot synthesize fructans, using the levansucrase-encoding ftf gene from S. mutans and the levansucrase-encoding SacB gene from B. subtilus is described in applications NL A 9300646 and WO 94/14970. The use of a levansucrase-encoding DNA sequence from Erwinia amylovora, which after integration in the host plant genome leads to the synthesis of levans, is described in DE 4227061 A1 and WO A 9404692. In all said applications, transgenic plants are described which are transformed with levansucrase genes from bacteria. Accordingly, these transgenic plants synthesize and accumulate fructans structurally comparable to those synthesized by the donor bacteria (Van der Meer et al., 1994; Ebskamp et al., 1994).
The present application differs from said applications in that it is related to fructosyltransferase-encoding DNA sequences derived from plants. These enzymes are structurally different from bacterial enzymes since there is no significant homology at the amino acid level and DNA level. Besides, the mechanism of fructan biosynthesis in plants is essentially different from that in bacteria. In contrast to fructan biosynthesis in bacteria, the formation of fructans in plants is mediated by more than one enzyme. For example, in Relianthus tuberosus (the Jerusalem Artichoke), fructan biosynthesis is catalysed by two enzymes: sucrose:sucrose fructosyltransferase (SST, EC 2.4.1.99) and fructan:fructan fructosyltransferase (FFT, EC 2.4.1.100). The SST and FFT from H. tuberosus are involved in the synthesis of .beta.-2,1 linked fructans (inulin) and are therefore also designated as 1-SST and 1-FFT. 1-FFT has been purified from tubers of H. tuberosus (Luscher et al., 1993, Koops and Jonker, 1994). The purification of SST has proven more difficult to achieve. A putative SST has been purified, at a very low yield, from several plant sources (Shiomi and Izawa, 1980; Praznik et al., 1990; Angenent et al., 1993). However, in none of these studies the purity of the enzyme has convincingly been shown. Furthermore, it has not conclusively been shown in these studies that the isolated enzyme does not represent an invertase.
Large quantities of 1-SST and 1-FFT have now been purified up to homogeneity from tubers of H. tuberogus (1-FFT: Koops and Jonker 1994) and their reaction mechanisms extensively investigated. 1-SST from H. tuberosus catalyses the initial step of fructan biosynthesis, the synthesis of the trisaccharide 1-kestose (1-[G-(F).sub.2 ]) from two molecules of sucrose (G-F), according to the following reaction: EQU G-F+G-F.fwdarw.1-[G-(F).sub.2 ]+G, (2)
wherein G-F=sucrose, -F=fructosylunit, -G=glucosylunit, G=glucose
1-SST can also catalyse the formation of the tetrasaccharide 1,1-[G-(F).sub.3 ] and pentasaccharide 1,1,1-[G-(F).sub.4 ] (FIG. 3A). Therefore, 1-SST activity can be described by the following general reaction: EQU G-(F).sub.n +G-(F).sub.m .fwdarw.G-(F).sub.n-1 +G-(F).sub.m+1, 1.ltoreq.n.ltoreq.3, 1.ltoreq.m.ltoreq.3 (3)
It has also been found that 1-SST from H. tuberosus to some extent can catalyse the transfer from a fructosyl unit from G-(F).sub.n, 1.ltoreq.n.ltoreq.3, onto water.
The second enzyme, 1-FFT, catalyses the formation of fructans with a higher degree of polymerization. This enzyme catalyses a polymerization reaction by the transfer of fructosyl units between trisaccharides, tetrasaccharides and larger fructose polymers according to the following general reaction: EQU G-(F).sub.n +G-(F).sub.m .fwdarw.G-(F).sub.n-1 +G-(F).sub.m+1, n.gtoreq.2, m.gtoreq.2 (4)
It has also been found that 1-FFT catalyses the transfer of fructosyl units between sucrose (G-F) and galactose (Gal)-containing carbohydrates [(Gal).sub.n -G-F], also called galactans. For example, 1-FFT can catalyse the transfer of a fructosyl unit from G-(F).sub.2 onto raffinose (Gal-G-F) which results in the formation of [Gal-G-(F).sub.2 ]. It cannot be excluded that both 1-SST and 1-FFT from H. tuberosus can use other substrates as fructosyl acceptor.
Although 1-SST and 1-FFT have some overlapping activity--both enzymes can catalyse the formation of tetra and pentasaccharides (reactions 3 or 4)--1-SST and 1-FFT are distinctly different enzymes. The 1-SST and 1-FFT proteins have different physical properties and are encoded by different genes. 1-SST and 1-FFT have essentially different enzymic properties. 1-FFT is not able to catalyse the initial step of fructan synthesis (reaction 2), whereas 1-SST is not able to catalyse the formation of fructan polymers with a degree of polymerization higher then 5 [G-(F).sub.n, n&gt;4]. In conclusion, with 1-SST activity alone, it is only possible to synthesize oligofructans from sucrose with a degree of polymerization of up to 5 [G-(F).sub.n, 2.ltoreq.n.ltoreq.4]. To synthesize fructans with a higher degree of polymerization and using sucrose as a substrate, both 1-SST and 1-FFT are needed. With 1-FFT activity alone, it is not possible to synthesize fructans from sucrose. It was found by the present inventors that protein fractions containing purified 1-SST as well as purified 1-FFT could use sucrose as a sole substrate for the synthesis of fructans with a degree of polymerization of at least 15 [G-(F).sub.14, FIG. 3B].
Bacterial fructans differ from fructans in plants with respect to the degree of polymerization and branching type and, consequently, in chemical and physical properties. In general, fructans from plants are assembled from less than 1000 fructosylunits. Fructans from H. tuberosus are assembled from less than 100 fruccosylunits. Fructans synthesized by bacteria may comprise more than 10.000 fructosyl units. Plant and bacterial fructans therefore differ in their possible applications. For fructans with a relatively low degree of polymerization, such as those isolated from Asteraceae (e.g. Jerusalem Artichoke, chicory or dahlia), an application as phosphate substitute in calcium binding agents and detergents has already been worked out (WO91/17189). Other applications are related to the organoleptical properties of fructans. The sweetening strength of fructans G-(F).sub.n decreases with an increasing degree of polymerization (increasing n-value). The sweetening strength of the oligofructans G-(F).sub.2 and G-(F).sub.3 approximates that of sucrose (G-F). The very long chain fructans such as those occurring in bacteria are not sweet at all. Very short chain fructans, such as those synthesized by sucrose:sucrose fructosyltransferase can therefore be used as sweeteners with the additional advantage that these sweet-tasting fructans are non-cariogenic and can withstand digestion in the digestive tract of humans, which opens possibilities for use as a low caloric sweetener. The short chain fructans, and also the longer chain fructans, can be used as the hydrophilic moiety of biosurfactants.
In contrast to the bacterial genes encoding levansucrase, which have already been cloned, the genes encoding SST and FFT have not been isolated before from plants. We found that the SST and FFT-encoding genes from plants, at the amino acid level, have no significant similarity to the known levansucrases and, at the DNA level, have no significant degree of homology to the levansucrase genes. For this reason it has not been possible to isolate the fructosyl transferase genes from plants using heterologous levansucrase probes from bacteria. It has also not been possible to isolate SST and FFT-encoding genes from plants using the amino acid sequences of the purified SST and FFT enzymes and their deduced oligonucleotide primers. The reason for this is that, although methods have been described for the purification of fructosyl transferases from plants, it has not been possible so far to obtain SST and FFT enzymes in sufficiently large amounts and with sufficiently high degrees of purity.